Pork longissimus muscles were used to study the effects of high pressure and specific temperatures on intramuscular fatty acid composition. After treatments at different pressures (300~700 MPa), temperatures (20~50 ℃), or treatment durations (10~20 min), the changes in the fatty acid composition of total lipids, triglycerides, phospholipids, and free fatty acids were observed. The results showed that pressure, followed by temperature, had the most significant impact on the fatty acid composition of pork. The interaction of pressure and temperature was significant, while the effect of treatment duration was no significant. There were mainly changes in the fatty acid composition of phospholipids and free fatty acids, whereas the fatty acid composition of intramuscular total lipids and triglycerides did not change significantly throughout the treatment process. There was significant degradation of phospholipids at pressures ≥300 MPa combined with thermal treatment. Additionally, more PUFA was degraded, resulting in a significantly decreased percentage of PUFA in phospholipids, while SFA and MUFA increased significantly in phospholipids. Moreover, the changes in the free fatty acid composition were the opposite of those observed in the phospholipid composition. Therefore, high-pressure and thermal treatment of pork mainly affected the fatty acid composition of phospholipids and free fatty acids but not of total intramuscular lipids and triglycerides.